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FungalGeneticsandBiology41(2004)973–981

/locate/yfgbi

Technologicaladvancement

Double-jointPCR:aPCR-basedmoleculartool

forgenemanipulationsinfilamentousfungi

Jae-HyukYu

a,

*

,1

,ZsuzsannaHamari

b,

*

,1

,Kap-HoonHan

a,1

,Jeong-AhSeo

a,1

,

´

nguez

b

,ClaudioScazzocchio

b,c

YazmidReyes-Domı

b

DepartmentofFoodMicrobiologyandToxicology,TheUniversityofWisconsin,Madison,WI53706,USA

ˆ

timent409,CentredÕOrsay,91405OrsayCedex,France

´

ne

´

tiqueetMicrobiologie,Universite

´

Paris-Sud,UMR8621CNRS,BaInstitutedeGe

c

InstitutUniversitairedeFrance,France

Received7July2004;accepted4August2004

a

Abstract

Genereplacementviahomologousdoublecrossoverinfilamentousfungirequiresrelativelylong(preferentially>0.5kb)flanking

sreason,generepla

facilitategenefunctionstudiesinfilamentousfungiavoidingtediouscloningsteps,wehavedevelopedaPCR-assistedDNAassem-

blyprocedureandappliedittodeletegenesinfiheprincipleofthisprocedureisessentiallythesameasother

recentlyreportedPCR-basedtools,ourtechniquehasbeeneffer,this

PCR-report,adetailedprotocolforthiseasy

tofollounctionwiththeavailabilityofgenome

sequences,theapplicationofthistechniqueshouldfacilitatefunctionalcharacterizationofgenesinfiamline

theanalysisofthetransformantsarelativelysimpleprocedureforgenomicDNAortotalRNAisolationachieving$100samples/

person/dayisalsopresented.

Óhtsreserved.

Keywords:PCR;Filamentousfungi;Genereplacement;Overexpression;Reporterfusion

uction

Theavailabilityofwholegenomesequencesfora

numberoffilamentousfungiincludingthemodelorgan-

ismsAspergillusnidulansandNeurosporacrassaaswell

asplantandhumanpathogensopensnewresearchave-

firststepofunderstandinggenefunctionsgen-

erallyinvolvesdisruptionand/orover-expressionof

inyeastwhereonlyabout

:+(J.-),

+33169157808().

E-mailaddresses:jyu1@(J.-),@

().

1

Theseauthorscontributedequally.

1087-1845/$-seefrontmatterÓhtsreserved.

doi:10.1016/.2004.08.001

*

50bpofhomologousDNAsequencesarerequiredfor

targetedintegration,genedisruptionbyhomologous

replacementinfilamentousfungiusuallyrequireslonger

DNAsequences(preferentially500bpormore).Thus,

constructionofgene-disruptioncassettesinfilamentous

fungiisusuallyaccomplishedthroughseveraltedious

fficient

functionalcharacterizationofgenesinfilamentousfungi

requiresasimpleandfastproceduretobuildgene-dis-

ruptionconstructs.

ThemethodofChaverocheetal.(2000)ensuresthe

presenceofverylongflankingsequences,butinvolves

alaboriousstepofinvivorecombinationinEscherichia

rast,PCR-basedfusiontechniquesobviate

cloningsteps(Davidsonetal.,2002;Kolkmanand

974J.-./FungalGeneticsandBiology41(2004)973–981

Stemmer,2001;Shevchuketal.,2004;Stemmer,

1994a,b)andPCR-assistedgenemanipulationswere

carriedoutinthepathogenicfungusCryptococcusneo-

formans(Davidsonetal.,2002).Independentlyfrom

thesestudies,wehavedevelopedaPCR-assistedtech-

niquethatcanbeusedtoconstructrecombinantDNA

moleculescombininganytwoorthreeDNAfragments.

Followingthistechnique,deletionof31geneswascar-

finalproductofeachdeletionconstruct

composedofthechosenselectivemakerwith0.5–

3.0kbupstreamanddownstreamfl

thisreport,wepresentadetailedprotocoltocarryout

thistechnique,whichwaseffectivelyusedforgene

manipulationsinthreefilamentousfungalspecies,A.

nidulans,Aspergillusfumigatus,andFusariumgraminea-

tion,simpleandfastmethodstoisolatefil-

amentousfungalgenomicDNAortotalRNAare

described.

alsandmethods:protocols

s,cultureconditions,genetics,and

transformation

nsstrainsderivedfromthestandardlab-

oratoryGlasgowstrainorFungalGeneticsStock

Centerandcarriedauxotrophicmarkersappropriate

ormation

recipientstrainsincludeRMS011(pabaA1,yA2;

DargB::trpC

+

;trpC801),PW1(biA1;argB2;methG1),

FGSC237(pabaA1,yA2;trpC801),CS2902(biA1,

pyrG89;pyroA4;riboB2),CS2903(biA1;wA3;pyroA4;

riboB2),CS2904(pabaA1,pyrG89;pantoB100),CS2905

(yA2,pantoB100,riboB2),CS2906(pyrG89,yA2;argB2;

pantoB100,riboB2).GeneticmarkersasinClutterbuck

(1994).tusstrainAF293.1(pyrG

À

)was

fromGregMayattheUniversityofTexas,-

earum(R-5317,equalto

MRC1781andNRRL5908)strainswerefromtheFusa-

riumResearchCenteratthePennStateUniversity.A.

nidulansmediaaredescribedinMartinelliandKinghorn

(1994).tus

wascarriedoutasdescribedbyTilburnetal.(1983)or

Hanetal.(2004a).earum

wasperformedaspreviouslydescribed(Leeetal.,2002).

jointPCR(DJ-PCR)tobuildagene

replacementconstruct

Atypicalreactionassemblingthreecomponents

nsasaselectivemarker

ral,sufficientamount

ofmakerDNAispre-amplified,cleaned,andstoredas

teDNAformarkergeneamplification

BgeneofA.

nidulansisamplifiedwiththefollowingprimers:argB-

For:5

0

-gaccagtttagaggcctc-3

0

,argB-Rev:5

0

-gtgtta

ggcctggatcta-3

0

.

oundPCR:amplificationof5

0

-and3

0

-

flankingregionsofthegene(s)ofinterest

(a)Designthefollowingsixprimersforthe1stround

PCR:5

0

-For,5

0

-Rev+markertail,3

0

-For+marker

tail,3

0

-Rev,5

0

-nestand3

0

-nest(seebelowandFig.1)

PCRmixture(final25ll)PCR

1llofpurified5

0

-flankingamplicon94°C2min

1llofpurified3

0

-flankingamplicon94°C30s—>

3llofpurifiedargBamplicon58°C10min·

10–15cycles

2llofdNTP(2.5mMeach)72°C5min—^

2.5llof10·PCRbuffer72°C10min

15.25llofsteriledistilledwater

0.25llofTaqpolymerase